The bacterial type III secretion system (T3SS) is a complex multi-protein apparatus that facilitates the secretion and translocation of effector proteins from the bacterial cytoplasm directly into the mammalian cytosol. This complex protein delivery device is shared by dozens of Gram-negative pathogens, including Salmonella spp., Shigella flexneri, Pseudomonas aeruginosa, Yersinia spp., enteropathogenic and enteroinvasive Escherichia coli, and Chlamydia spp. These pathogens collectively cause over 200 million cases of human illness and greater than half a million deaths worldwide each year. They are the causative agents of plague, pneumonia, typhoid fever, and other diseases that impact human health. See, e.g., www.who.int; Morris and Potter, Eds., Foodborne Infections and Intoxications, Academic Press, New York, 4th ed. (2013); Cornelius, Nat. Rev. Microbiol. 4: 811-825 (2006). The issue of antibiotic resistance is most pressing for Gram-negative bacteria, for which only one new class of antibiotics has been approved in the last 15 years. Projan et al., Curr. Opin. Microbiol. 10: 441-446 (2007); Boucher et al., Clin. Infect. Dis. 56: 1685-1694 (2013).
The T3SS is composed of a basal structure spanning the inner and outer bacterial membranes and a needle that extends from the bacterial surface. Moraes et al., Curr. Opin. Struct. Biol. 18: 258-266 (2008). This structure acts as a molecular syringe that injects bacterial effector proteins directly inside target host cells. While the structure of the T3SS is relatively conserved among T3SS-expressing bacteria, the suite of T3SS effector proteins expressed by each group of pathogens is completely distinct. Cornelius, Nat. Rev. Microbiol. 4: 811-825 (2006). The Yersinia pseudotuberculosis T3SS has been extensively studied and is often used as a model for T3SS-expressing pathogens. Duncan et al., Antimicrob. Agents Chemother. 56: 5433-5441 (2014), which is hereby incorporated by reference in its entirety. In Yersinia, the T3SS translocon proteins, LcrV, YopB, and YopD, form a pore in the mammalian plasma membrane upon host cell contact, and enable translocation of effector proteins inside the host cell cytosol. Bleves et al., Microbes Infect. 2: 1451-1460 (2000). Y. pseudotuberculosis effector proteins YopH, YopO, YopT, and YopE block phagocytosis and the formation of reactive oxygen species, while YopJ, YopM, and YopK dampen innate immune signaling. Bliska et al., Cell Microbiol. (2013); Clatworthy et al., Nat. Chem. Biol. 3: 541-548 (2007).
Although the bacterial T3SS is a broadly distributed apparatus important for disease causation, the precise mechanism of type III secretion remains incompletely understood. Thus, there remains a need to develop screening methods for identifying potent inhibitors of T3SSs in various bacterial species, including Yersinia pseudotuberculosis. 